Project Summary Autografts and allografts are current standard strategies for bone repair of oral and craniofacial defects. However, each possesses limitations, such as donor-site morbidity with the use of autograft and the risk of dis- ease transmission with the use of allograft. Synthetic bone-graft substitute based on a tissue engineering strat- egy has represented an alternative approach to overcome these inherent limitations. MicroRNAs (miRs) are small non-coding RNAs that have emerged as important transcriptional regulators in both physiologic and pathophysiological conditions. We have previously shown that members of the miR-200 family actively regulate osteogenic differentiation, bone development, and inflammation. Our long-term goal is to develop a miR-based gene therapy program that can be used to effectively promote osteogenic differentiation and bone regeneration for restoring oral and craniofacial bone defects. Our recent studies showed that miR-200c loaded in collagen sponge promoted bone formation in vivo. We also observed that miR-200c up-regulated the activity of Wnt sig- naling and reduced Sox2 and Klf4. Relatedly, miR-200a has been demonstrated to directly suppress Wnt and BMP/TGF signal pathways. In our preliminary studies we have shown that inhibiting miR-200a using our Plas- mid-based miR Inhibitor System (PMIS) can effectively improve osteogenic differentiation and bone formation in vivo. Our objectives in this application are to understand and optimize the molecular function of miR-200c and PMIS-200a (miR-200a inhibitor) on osteogenic differentiation and bone formation and to validate that their local administration can be used to improve bone regeneration. The central hypothesis of this project is that miR-200c combined with miR-200a inhibition improve osteogenic differentiation by up-regulating Wnt and TGF- /BMP signaling. Plasmid miR-200c and PMIS-200a delivery can be optimized to promote bone regeneration for oral and craniofacial defects. In this project, we will determine the molecular function of miR-200c on Wnt sig- naling and the roles of Sox2 and Klf4 in the osteogenic differentiation mediated by miR-200c (Aim 1). We will also determine the function of PMIS-200a on osteogenic differentiation and bone formation by understanding the regulation of PMIS-200a on Wnt and BMP/TGF signaling (Aim 2). We will improve the effectiveness of miR-200c and PMIS-200a by optimizing a gene delivery system using biodegradable nanoparticles. In the Aim 3 we will use critical-sized calvarial defects and periodontitis-induced bone defects in rat models to investigate whether miR-200c combined with PMIS-200a can induce significant bone regeneration in vivo. At the comple- tion of this project, we will have significantly expanded our understanding of the molecular function of miR-200c and miR-200a inhibition and demonstrated the translational capabilities of these miRs for oral and craniofacial bone regeneration.